INTERACTION OF THE MAMMALIAN ANTIBACTERIAL PEPTIDE CECROPIN P1 WITH PHOSPHOLIPID-VESICLES

Cecropins are positively charged antibacterial polypeptides that were originally isolated from insects. Later on a mammalian homologue, cecr opin P1 (CecP), was isolated from pig intestines. While insect cecropi ns are highly potent against both Gram-negative and Gram-positive bact eria, CecP is as active as insect cecropins against Gram-negative but has reduced activity against Gram-positive bacteria. To gain insight i nto the mechanism of action of CecP and the molecular basis of its ant ibacterial specificity, the peptide and its proline incorporated analo gue (at the conserved position found in insect cecropins), P-22-CecP, were synthesized and labeled on their N-terminal amino acids with fluo rescent probes, without significantly affecting their antibacterial ac tivities. Fluorescence studies indicated that the N-terminal of CecP i s located on the surface of phospholipid membranes. Binding experiment s revealed that CecP binds acidic phosphatidylserine/phosphatidylcholi ne (PS/PC) vesicles better than zwitterionic PC vesicles, which correl ates with its ability to permeate the former better than the latter. T he shape of the binding isotherms suggest that CecP, like insect cecro pin, binds phospholipids in a simple, noncooperative manner. However, resonance energy transfer (RET) measurements revealed that, unlike ins ect cecropins, CecP does not aggregate in the membrane even at relativ ely high peptide to lipid ratios. The stoichiometry of CecP binding to vesicles suggests that amount of CecP sufficient to form a monolayer causes vesicle permeation. In spite of the incorporation of the conser ved proline in P-22-CecP, the analogue has reduced antibacterial activ ity, which correlates with its reduced alpha-helical structure and its lower partitioning and membrane permeating activity with phospholipid vesicles. Taken together, our results support a mechanism in which Ce cP disrupts the structure of the bacterial membrane by (i) binding of peptide monomers to the acidic surface of the bacterial membrane and ( ii) disintegrating the bacterial membrane by disrupting the lipid pack ing in the bilayers. These results, combined with data reported for ot her antibacterial polypeptides, suggest that the organization of pepti de monomers within phospholipid membranes contributes to Gram-positive /Gram-negative antibacterial specificity.